PROJECT SUMMARY/ABSTRACT - PROJECT 2 Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS) associated with increases in activated myelin-reactive T cells. We have previously identified immunoregulatory defects in patients including loss of CD4+Foxp3+ regulatory T cells (Tregs), and a decrease in expression of TIM-3 on CD4 T cells. TIM-3 is selectively expressed on Th1 cells, and engagement of TIM-3 by its ligands galectin-9 or the recently discovered CEACAM-1 regulates Th1 cells and controls tolerance induction. Blocking the TIM-3/CEACAM-1 pathway exacerbates disease in a mouse model for MS. The mechanisms underlying the loss of TIM-3 in MS are not known. We recently undertook a transcriptional profiling study from PBMC of MS patients and identified two subsets of MS patients (MSA and MSB), with MSA patients showing a more active disease. Our preliminary data indicate that lower expression of TIM-3 distinguished the MSA group. Furthermore, a beneficial response to interferon-? (IFN?) therapy correlated with the degree that TIM-3 was upregulated. Additional experiments identified IFN? and IL-27, which can be induced by IFN? in APCs, as major inducers of TIM-3 on T cells. Taken together with recent reports showing low IFN? serum levels in patients, this suggests that a defect in the IFN?-TIM-3 pathway may play a part in the development of MS. Moreover, our preliminary studies show that TIM-3 is upregulated on human Tregs after activation and that TIM-3+ Tregs are more efficient suppressors than TIM-3- Tregs. Thus, the previously reported loss of Treg function in MS patients that disappeared with IFN? treatment may be linked to defective TIM-3 expression. Our overall hypothesis is that a genetically mediated loss of type I IFN signal in MS drives a loss of TIM-3 expression and of immunoregulation. We propose studies in both humans and mouse models to accomplish the following aims: 1. To determine the mechanism by which IFN? and IL-27 regulate TIM-3 expression and IL-10 secretion by effector T cells. 2. To define the role of TIM-3+ Tregs in regulating autoimmunity in the CNS. These investigations provide a link between the genetic variations underlying autoimmune disease with functional alterations in immune function.